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  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • G—PHYSICS
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  • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
  • G01N2333/4701—Details
  • G01N2333/4748—Details p53

Definitions

• the present invention relates to the field of cellular senescence and cancer therapy, more particularly to combination therapies and use thereof for the treatment of cancer.
• combinations comprising a CDC7 inhibitor and a mTOR inhibitor and/or an immune checkpoint inhibitor, and compositions and kits comprising such inhibitor of CDC7 and/or such inhibitor of mTOR and/or such immune checkpoint inhibitor, for use in the treatment of cancers including, but not limited to liver cancer, lung cancer, non-small lung cancer, colon cancer.
• Cancer is a leading cause of death worldwide, accounting for more than 8.8 million deaths in 2015. Generally, when cancer develops, normal cells become progressively abnormal over time as they acquire mutations, which allow them to escape immune surveillance, survive, grow uncontrollably, and spread through the body (Lazebnik, Y., (2010), Nature Reviews Cancer, Vol. 10, pages 232-233; Bekele and Brindley (2012), Clinical Lipidology, Vol.7, pages 313-328).
• cetuximab is approved for use in colon cancer and is beneficial, but adding it to another effective regimen (capecitabine, oxaliplatin and bevacizumab) did not improve therapy.
• Senescence is a physiological process of (essentially)“stable cell-cycle arrest”, which leads to the loss of proliferative (cell division) capacity of mitotic cells (cells having the ability to divide), even in the presence of mitogenic signals. Senescence plays a role in natural aging. Although senescent cells have lost their ability to proliferate, they remain viable and metabolically active (e.g. capable of secretary activity such as secreting cytokines, growth factors and proteases, and others), a phenotype referred to as senescence-associated secretory phenotype.
• cancer cells have the ability to overcome therapy- induced senescence resulting in the cancer cells (also known as senescence escapers) being able to resume proliferation (Sabine and Anderson (201 1), Genome Integrity, Vol.2(1), page 7; Nair et al (2017), Biomedical Research Journal, Vol. 4(1 ), pages 28-48; Munoz-Espin et al (2014), Nat Rev Mol Cell Biol, Vol.15(7), pages 482- 96).
• senescent cancer cells e.g. senescence escaper cancer cells
• paracrine-active factors capable of influencing growth and migration of cells in the tumor environment (e.g. promote growth of epithelial cells) and other factors promoting inflammation
• paracrine-active factors capable of influencing growth and migration of cells in the tumor environment
• other factors e.g. promote growth of epithelial cells
• BMB reports Vol. 47(2), pages 51-59
• the present inventors have uncovered a new cancer therapy relying on the concept of therapy-induced senescence and which is devoid of at least some of the limitations current associated with such therapies.
• a new combination therapy comprising (the use of) a CDC7 inhibitor and an mTOR inhibitor.
• the therapy with the CDC7 inhibitor and the mTOR inhibitor further includes treatment with an immune checkpoint inhibitor.
• a combination therapy comprising (the use of) a CDC7 inhibitor and an immune checkpoint inhibitor.
• new cancer treatment methods including methods to identify a cancer subject suitable for treatment with the combination therapy of the invention.
• this particular combination of compounds i.e. a CDC7 inhibitor and a mTOR inhibitor
• cancer including, but not limited to, liver cancer, lung cancer, non-small lung cancer, and colon cancer, both in vitro and in vivo, as shown herein.
• This effect was not seen when other senescence inducing drugs, not a CDC7 inhibitor, were used.
• the cancer growth inhibition effect is the consequence of induction of senescence by specifically inhibiting CDC7 in the cancer cell using a CDC7 inhibitor, as exemplified by XL413, Tak-931 , and LY3177833 and the killing of these specific cells (e.g. via apoptosis) in response to exposure to a mTOR inhibitor, as exemplified by AZD8055 or AZD2014.
• a mTOR inhibitor as exemplified by AZD8055 or AZD2014.
• the observed effect is not limited to those combinations of CDC7 inhibitors and mTOR inhibitors tested in the Examples herein.
• the data supports the surprising conclusion that it is the inhibition of CDC7 and the inhibition of mTOR in cancer cells that provides for the observed beneficial effects.
• the CDC7 inhibitor is XL413 and the mTOR inhibitor is AZD8055;
• the CDC7 inhibitor is TAK-931 and the mTOR inhibitor is AZD8055;
• the CDC7 inhibitor is LY3177833 and the mTOR inhibitor is AZD8055;
• the CDC7 inhibitor is LY3177833 and the mTOR inhibitor is AZD2014;
• the CDC7 inhibitor is TAK-931 and the mTOR inhibitor is AZD2014;
• the CDC7 inhibitor is XL413 and the mTOR inhibitor is AZD2014.
• the combination therapy of the present invention offers several advantages over existing cancer therapies
• a CDC7 inhibitor such as XL413, or TAK931 or LY3177833
• mTOR inhibitor such as AZD8055 or AZD2014
• the combination selectively acts on or targets cancer cells, more in particular the mTOR inhibitor selectively acts on or target cancer cells treated with a CDC7 inhibitor.
• the invention provides for a new way of using an mTOR inhibitor in the treatment of cancer by treating cancer cells treated with a CDC7 inhibitor.
• the invention provides for a new way of using a CDC7 inhibitor in the treatment of cancer by treating cancer cells treated with a mTOR inhibitor.
• Another advantage of the combination therapy is that it allows to rid the body of senescent cancer cells artificially induced to be senescent by exposure to a CDC7 inhibitor before the senescent cancer cells can exit the senescent fate (e.g. by activating senescence reversal mechanisms) and/or before the senescent cells can exert harmful effects having negative influences on surrounding (healthy) cells and tissues (e.g. secretion of paracrine signals and /or pro-inflammatory molecules, etc.).
• the combination therapy of the invention not only effectively stops or slows cancer cell growth or tumor growth but also specifically removes the targeted cancer cells. This can result in enduring, long-term beneficial effects in cancer subjects (such as mammals, e.g. human beings) receiving or being administered with the combination as taught herein, such as decrease in tumor size and/or increased survival (increased progression-free survival), as well as increased overall (positive) response rate in cancer subjects.
• cancer subjects such as mammals, e.g. human beings
• the combination as taught herein such as decrease in tumor size and/or increased survival (increased progression-free survival), as well as increased overall (positive) response rate in cancer subjects.
• the present combination was found to be particularly effective for treating cancer subjects wherein the cancer is characterized by a mutated p53 gene, i.e. wherein the patient carries mutations in the p53 gene relative to wild-type p53, for example loss-of-function mutations or other defects leading to the occurrence of non-functional p53 gene or protein of altered function. Therefore, the present combination therapy represents a new effective treatment strategy which is devoid of at least some of the disadvantages of existing cancer therapies as discussed above.
• experimental results provide for a combination therapy with a combination of a CDC7 inhibitor and an immune checkpoint inhibitor.
• This combination may, in a preferred embodiment, further include treatment with an mTOR inhibitor.
• experimental data indicates that liver tumors induced by loss of Tp53 and overexpression of the Myc oncogene in immunocompetent animals undergo senescence in vivo after treatment with CDC7 inhibitor.
• the data also revealed that tumors from these animals are infiltrated with CD8-positive T cells that are also Ki67 positive, a marker of proliferation.
• CD8-positive T cells that are also Ki67 positive, a marker of proliferation.
• it was found that these T cells are unable to kill the senescent tumor cells, as judged by a lack of decline in senescent cells in vivo over time.
• the present inventors have devised new treatment methods relying on the use of the combination of the invention (a CDC7 inhibitor and a mTOR inhibitor or a CDC7 inhibitor and an immune checkpoint inhibitor or a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor).
• the combination therapy of the invention is particularly beneficial or effective in the treatment of cancer (e.g. liver cancers) having a mutated p53 gene, e.g. causing a I oss-of-f unction of the p53 protein thus providing a“personalized” treatment or medicine to a particular cancer patient group, wherein the cancer patients are characterized by having (e.g. in their genome) a p53 mutation (e.g. p53 mutant genotype or phenotype) or other genetic or metabolic alterations leading to the occurrence of non-functional p53 gene product (p53 proteins).
• cancer e.g. liver cancers
• a mutated p53 gene e.g. causing a I oss-of-f unction of the p53 protein thus providing a“personalized” treatment or medicine to a particular cancer patient group
• the cancer patients are characterized by having (e.g. in their genome) a p53 mutation (e.g. p53 mutant genotype or phen
• a portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction).
• copyright protection such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.
• the copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.
• a method for administrating a drug includes the administrating of a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).
• the term“and/or” indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.
• the term "to comprise” and its conjugations as used herein is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting“to consist of”.”
• cancer refers to or describe the physiological condition in humans that is typically characterized by unregulated cell growth.
• the terms“cancer” and“tumor” also refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Cancer cells can be distinguished from non-cancerous cells by techniques known to the skilled person.
• subject or“patient” (used interchangeably) as used herein refers to a human subject male or female, adult, child or infant, suffering from a cancer, regardless of the stage or state of the cancer.
• treat refers to reducing or ameliorating a disorder (herein cancer) and/or symptoms associated therewith. It is appreciated that treating a disorder or condition (e.g. cancer such as lung cancer) does not require that the disorder, condition or symptoms associated therewith be completely eliminated. It is further understood that the terms “treat,” treating”, “treatment”, “therapy” and as used herein may be a first or first line of treatment (i.e. patient is naive to any cancer treatment) or a second or third line treatment and so on (i.e. the first treatment or second treatment and so on was not effective or has failed).
• first or first line of treatment i.e. patient is naive to any cancer treatment
• second or third line treatment and so on i.e. the first treatment or second treatment and so on was not effective or has failed.
• inhibitor of a (defined) protein refers to a compound capable of down-regulating, decreasing, suppressing gene expression and/or protein production or otherwise regulating the amount and/or activity (e.g. enzymatic activity) of the (defined) gene or protein (i.e. CDC7 or mTOR, or an immune checkpoint protein such as PD-1 , PD-L1 or CTLA-4 as taught herein).
• the compound inhibits the activity (e.g. enzymatic activity) of CDC7 or mTOR or of the immune checkpoint protein such as PD-1 , PD-L1 or CTLA-4.
• Such compounds are well-known to the skilled person and are generally referred to in the prior art as CDC7 inhibitor or mTOR inhibitor or immune checkpoint inhibitor (e.g. PD-1 inhibitor, PD-L1 inhibitor or CTLA- 4 inhibitor).
• CDC7 inhibitor or mTOR inhibitor or immune checkpoint inhibitor e.g. PD-1 inhibitor, PD-L1 inhibitor or CTLA- 4 inhibitor.
• immune checkpoint inhibitor e.g. PD-1 inhibitor, PD-L1 inhibitor or CTLA- 4 inhibitor.
• marketing authorizations and drug approvals identify approved drugs not only by structure but also by its mode of action, in this case by identifying a compound as a CDC7 inhibitor and/or as mTOR inhibitor and/or an immune checkpoint inhibitor, reflecting the understanding by the skilled person that the treatment is based on the inhibition of, in the current case, CDC7, mTOR or the relevant immune checkpoint (e.g. PD-L1 , PD-1 or CTLA-4).
• (candidate) drugs are studied by its virtue of being a modulator of enzyme activity, for example by being a CDC7 inhibitor or a mTOR inhibitor or an immune checkpoint inhibitor. Consequently, a skilled person very well understands what, in the context of the current invention is an inhibitor of CDC7. Consequently, a skilled person very well understands what, in the context of the current invention is an inhibitor of mTOR. Consequently, a skilled person very well understands what, in the context of the current invention is an immune checkpoint inhibitor, e.g. a PD-L1 inhibitor, a PD-1 inhibitor or a CTLA-4 inhibitor.
• an immune checkpoint inhibitor e.g. a PD-L1 inhibitor, a PD-1 inhibitor or a CTLA-4 inhibitor.
• CDC7 cell division cycle 7-related protein kinase
• CDC7 refers to an enzyme involved in regulation of the cell cycle at the point of chromosomal DNA replication.
• CDC7 is encoded by the CDC7 gene (ensemble ref: ENSG00000097046).
• CDC7 (protein) is predominantly localized in the nucleus and serves as a cell division cycle protein with kinase activity.
• CDC7 (protein) is a serine-threonine kinase that plays a key role in the initiation of DNA replication and regulation of the S phase cell cycle check point.
• CDC7 activity requires the binding of either one of two regulatory subunits, Dbf4 and Drf1/Dbf4B.
• CDC7 inhibitor As used herein is well-understood by the skilled person and refers to compounds or drugs (e.g.
• small molecules capable of, in particular in vivo, for example when provided to a patient, (specifically) down-regulating, decreasing, suppressing or blocking the expression of the CDC7 gene and/or down regulating, decreasing, suppressing or blocking the production of its product (CDC7 protein), or otherwise (specifically) regulating (e.g. by decreasing or blocking or suppressing) the amount and/or activity (e.g. enzymatic activity) of CDC7 kinase.
• the CDC7 inhibitor inhibits the activity of the CDC7 kinase.
• a CDC7 inhibitor compound is a compound capable of (specifically) inhibiting CDC7 by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% or more compared to control.
• the CDC7 inhibitor is a compound capable of (specifically) inhibiting CDC7 comparable to XL413 (e.g. 0.1 - 10 fold potency).
• CDC7 activity inhibition can be measured, for example, as described in EP2970221.
• CDC7 inhibitors e.g. CDC7 kinase ATP-competitive small- molecule inhibitors belonging to the 2-heteroaryl-pyrrolopyridones chemical class
• nerveiano Medical Sciences Sri e.g. see Vanotti et al (2008), J Med Chem, Vol. 51 , pages 487-501 ; Montagnoli et al (2010) American Association of Cancer Research, Vol. 16(18), pages 4503-4508
• Other CDC7 inhibitors include NMS-1 1 16354 (Montagnoli et al (2010) American Association of Cancer Research, Vol.
• XL-413 also known as BMS-863233 from Bristol Myers Squibb (BMS), e.g. see Koltun et al (2012), Bioorg Med Chem Lett., Vol. 22(1 1 ), pages 3727-31 ; Montagnoli et al (2010) American Association of Cancer Research, Vol. 16(18), pages 4503-4508), and SRA141 (Sierra Oncology, e.g. Montagnoli et al (2010) American Association of Cancer Research, Vol. 16(18), pages 4503-4508).
• CDC7 inhibitors include TAK-931 (Takeda oncology), LY3177833 as well as compounds described in W02005/100351 , W02014143601A1 , and in Masaaki Sawa and Hisao Masai (2008), Drug design, Development and Therapy, Vol. 2, pages 255- 264).
• mTOR mammalian target of rapamycin
• mTOR refers to a kinase, which is a member of the phosphatidylinositol 3-kinase-related kinase family of protein kinases.
• mTOR is encoded by the MTOR gene (Ensembl ref: ENSG00000198793).
• mTOR is also known as“the mechanistic target of rapamycin and FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1)”. mTOR links with other proteins and serves as a core component of two distinct protein complexes, mTOR complex 1 and mTOR complex 2, which regulate different cellular processes. In particular, as a core component of both complexes, mTOR functions as a serine/threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. As a core component of mTORC2, mTOR also functions as a tyrosine protein kinase that promotes the activation of insulin receptors and insulin-like growth factor 1 receptors.
• FRAP1 12-rapamycin-associated protein 1
• mTORC2 has also been implicated in the control and maintenance of the actin cytoskeleton.
• Interest in mTOR as a potential cancer therapy target came from studies on rapamycin. Rapamycin is one of the first mTOR inhibitors, which was developed as an antifungal drug against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. It was later discovered that rapamycin also had anti-cancer activity against several human cancer types (e.g. see Faivre et al (2006), Nature Reviews Drug Discovery, Vol. 5 (8). pages 671-88). This prompted the development of several mTOR inhibitors (e.g.
• rapamycn derivatives or analogues such as temsirolimus (CCI-779, Wyeth), everolimus (RAD001 , Novartis), and ridaforolimus (AP-235730, Ariad Pharmaceuticals) (see e.g. Brachman et al (2009), Current Opinion in Cell Biology, Vol.21 (2), pages 194-8). Rapamycin and its analogues only inhibit mTOR1.
• Other non-limiting examples of mTOR inhibitor compounds are listed below. However, it was found that in general, the development of mTOR inhibitors for the treatment of cancer (e.g.
• mTOR inhibitor as used herein is well-understood by the skilled person and refers to compounds or drugs (e.g. small molecules) capable of, in particular in vivo, for example when provided to a patients, (specifically) down-regulating, decreasing, suppressing or blocking the expression of the mTOR gene and/or down regulating, decreasing, suppressing or blocking the production of its product (mTOR protein) or otherwise (specifically) regulating (e.g. by decreasing or blocking or suppressing) the amount and/or activity (e.g. enzymatic activity) of mTOR kinase
• the CDC7 inhibitor inhibits the activity of the CDC7 kinase.
• a mTOR inhibitor compound is a compound capable of (specifically) inhibiting mTOR by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% or more as compared to control.
• the mTOR inhibitor is a compound capable of (specifically) inhibiting mTOR comparable to AZD8055 (e.g. with a 0.1 - 10 fold potency).
• AZD8055 e.g. with a 0.1 - 10 fold potency
• Non-limiting examples of mTOR inhibitors include AZD8055 (e.g. see Chresta et al, (2010) Cancer Res., Vol. 70(1), pages 288-98), and AZD2014 (AstraZeneca ,e.g. see (2015), Guichard et al (2015) Mol Cancer Ther., Vol. 14(1 1), pages 2508-18), TAK931 and LY3177833.
• the mTOR inhibitors preferably inhibit mTOR2 (see for selectivity of mTOR inhibitors Cancer Res. 2010 Jan 1 ; 70(1):288-98.).
• the mTOR inhibitors preferably inhibit both the mTOR complexes, i.e. inhibit both mTOR 1 and mTOR2. It is noted that, for example, rapamycin, which only inhibits mTOR1 was way less active in our assays as compared to the mTOR inhibitors that are also inhibitors of mTOR2.
• Immune checkpoint inhibitor is well-understood by the skilled person. Immune checkpoints are proteins and regulators of the immune system and are crucial for self-tolerance, which prevents the immune system from attacking cells indiscriminately. Inhibitory checkpoint molecules are targets for cancer immunotherapy due to their potential for use in multiple types of cancers.
• Immune checkpoint inhibitors include inhibitors of CTLA4 (cytotoxic T lymphocyte antigen-4), PD-1 (programmed cell death protein 1), PD-L1 (programmed cell death ligand 1), PD-L2 (programmed cell death ligand 2), PD-L3 (programmed cell death ligand 3), PD- L4 (programmed cell death ligand 4), LAG-3 (lymphocyte activation gene-3), and TIM-3 (T cell immunoglobulin and mucin protein-3).
• the immune checkpoint inhibitors are inhibitors of PD-1 , PD-L1 or CTLA-4.
• the immune checkpoint inhibitor is a binding ligand of PD-1 or a binding ligand of PD-L1.
• the immune checkpoint inhibitor is a binding ligand of CTLA-4.
• the immune checkpoint inhibitor is an antibody, e.g. an antibody for PD-1 , PD-L1 or CTLA-4.
• PD-1 is a key immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression.
• PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 , and BTLA.
• the term "PD-1” as used herein includes (human) PD-1 , variants, isoforms, and species homologs of human PD-1 , and analogs having at least one common epitope with human PD-1.
• PD-L1 Various cell surface glycoprotein ligands for PD-1 have been identified, including PD-L1 , PD- L2, PD-L3, and PD-L4, that are expressed on antigen-presenting cells as well as many human cancers and have been shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 .
• the term "PD-L1” as used herein includes human PD-L1 (hPD-L1 ), variants, isoforms, and species homologs of human PD-L1 , and analogs having at least one common epitope with human PD-L1.
• CTLA-4 cytotoxic T-lymphocyte-associated protein 4
• CTLA-4 cytotoxic T-lymphocyte-associated protein 4
• CTLA4 is found on the surface of T cells, is also a member of the immunoglobulin (Ig) superfamily; CTLA-4 comprises a single extracellular Ig domain.
• CTLA-4 transcripts have been found in T cell populations having cytotoxic activity, suggesting that CTLA-4 might function in the cytolytic response.
• CTLA-4" as used herein includes human CTLA-4, variants, isoforms, and species homologs of human CTLA-4I, and analogs having at least one common epitope with human CTLA-4.
• inhibitors of PD-1 include nivolumab (Opdivo®, Bristol-Myers Squibb), pembrolizumab (Keytruda®, Merck), BGB-A317, and others such as PDR001 (Novartis).
• Other non-limiting examples of PD-1 antagonists include pidilizumab (Cure Tech), AMP-224 (GlaxoSmithKline), AMP-514 (GlaxoSmithKline), PDR001 (Novartis), and cemiplimab (Regeneron and Sanofi).
• inhibitors of PD-L1 includes atezolizumab (Tecentriq) from Roche, Avelumab (Bavencio) developed by Merck Serono and Pfizer and Durvalumab (Imfinzi) developed by AstraZeneca.
• inhibitors of CTLA-4 include antagonistic antibodies against CTLA-4 such as ipilimumab ((Yervoy®, MDX-010, Bristol-Myers Squibb)
• tumor protein 53 (abbreviated as p53)” as used herein is well-known to the skilled person and refers to a nucleophosphoprotein that binds DNA and regulates normal cell growth and proliferation and prevents unrestrained division of cells whose DNA has been damaged (e.g. via ultraviolet or ionizing radiation).
• p53 tumor protein 53
• P53 is encoded by the TP53 gene (humans, ensemble ref: ENSG0000014151). Therefore, the term "TP53" or“p53 gene” as used herein refers to the gene encoding p53 protein.
• p53 is also known as cellular tumor antigen p53, phosphoprotein p53, tumor suppressor p53, antigen NY- CO-13, or transformation-related protein 53 (TRP53) or any isoform of a protein encoded by homologous genes in various organisms
• TRP53 transformation-related protein 53
• mutated p53 gene or“p53 mutation” as used herein refers to genetic mutation(s) in the tumor protein 53 (TP53) gene.
• “mutated p53 gene” or“p53 mutation” refers to any p53 protein or gene other than wild- type p53 protein or gene. It was found that the TP53 gene is one of the most commonly mutated gene in human cancer (Muller and Vousden (2014), Cancer cell, Vol. 25, pages 304-317). Mutations in the TP53 gene typically lead to a loss of wild-type p53 activity (i.e.
• Alterations of a wild-type p53 gene according to the present invention encompass all forms of mutations such as insertions, inversions, deletions, and/or point mutations and includes somatic and germ line mutations and that cause a loss-of-function. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germ line. Germ line mutations can be found in any of a body's tissues. As previously described 70% of TP53 mutations are missense mutations affecting residues within the p53 DNA-binding domain (DBD).
• DBD p53 DNA-binding domain
• administered or“administering” or“providing” as taught herein refers to the act of providing or administering a subject with the combination of the invention i.e. , a CDC7 inhibitor compound and a mTOR inhibitor compound and/or an immune checkpoint inhibitor, which can be administered to said subject simultaneously, separately or sequentially (as explained herein), using any of the various methods of delivery systems well known to those skilled in the art.
• the administering can be performed, for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, introccularly, via local delivery, subcutaneously, intraadisposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventricularly, intratumorally, into cerebral parenchyma or intraparenchymally or microinjection or others.
• parenterally intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, introccularly, via local delivery, subcutaneously, intraadisposally, intraarticularly, intrathecally, into a cerebral ventricle, intraventricularly, intratumorally, into cerebral par
• sensescence refers to a phenomenon by which normal cells stop or cease to divide. Senescence was originally identified through the limited ability of primary fibroblasts in culture to undergo cell division (Hayflick et al (1965), Exp. Cell Res, Vol.37, pages 614-636).
• senescence a stable state of growth arrest
• SA-b- gal senescence associated b-galactosidase
• SAHFs senescence-associated heterochromatin foci
• Senescence is generally considered to be a fail-safe mechanism against oncogenic transformation, as expression of an oncogenic RAS gene in primary cells leads to a post-replicative state referred to as oncogene- induced senescence (Serrano et al (1997), Cell, Vol.88 (5), pages 593-602).
• This fail-safe mechanism also operates in humans to prevent cancer, as melanocytic nevi (moles) often carry an activated BRAF(V600E) oncogene, but stain for many of the known senescence markers, indicative of a stable and lasting state of oncogene- induced senescence in these cells (Michaloglou et al (2005), Nature, Vol.436(7051), pages 720-724).
• Senescent cells also secrete a variety of inflammatory cytokines and chemokines, collectively referred to as the“Senescence-Associated Secretory Phenotype (SASP) (Kuilman et al (2008), Cell, Vol.133(6), pages 1019-1031 ; Coppe et al (2008), PLoS Biol, Vol. 6(12), pages 2853-2868).
• SASP Session-Associated Secretory Phenotype
• senescent cells or“senescent cancer or tumor cells” as used herein refers to cells (e.g. cancer cells), which are in a state of growth arrest.
• a cell capable of dividing may become senescent (in a naturally-occurring way, i.e. not induced by a drug compound) when, for example, it encounters oncogenic stress or undergo DNA damage (e.g. see Rodier F (201 1) J Cell Biol. 201 1 , DOI: 10.1083/jcb.201009094, from which the description of senescence is incorporated herein by reference).
• the term "senescent tumor cells” or “senescent cancer cells” encompasses cancer cells which have adopted the cellular senescence phenotype, for example as evidenced by growth arrest and/or by the presence of a marker or combination of markers that are characteristic of senescence.
• markers include but are not necessarily limited to the pl6INK4a tumor- suppressor protein, and modified, e.g. increased, expression relative to a reference, such as a non-senescent cell, in the levels of DNA-damage response (DDR) markers, as well as the cell cycle inhibitors pl6INK4A, pl5INK4B, p21 CIP1 , and p53.
• senescent cells are SA-beta-Gal (senescence-associated beta galactosidase) positive.
• combination therapy or "in combination with” as used herein is intended to refer to all forms of administration that provide a first drug (e.g. a CDC7 inhibitor compound as taught herein) together with a further (second, e.g. a mTOR inhibitor compound as taught herein and/or an immune checkpoint inhibitor as taught herein).
• first drug e.g. a CDC7 inhibitor compound as taught herein
• second e.g. a mTOR inhibitor compound as taught herein and/or an immune checkpoint inhibitor as taught herein
• the separate drugs may be administered simultaneously, separately or sequentially, and in any order.
• Drugs administered in combination have biological activity in the subject to which the drugs are delivered, and more particular the combination has biological activity in the same cancer cell.
• the drugs are administered or provided separately or sequentially (one after the other, in any order), it is preferable that the drugs are administered or provided within a certain time limit such that the pharmacological effect of the first drug is still noticeable in the patient, more in particular in the cancer cells in the patient.
• the treatment is simultaneously, i.e. wherein the CDC7 inhibitor and the mTOR inhibitor and/or immune checkpoint inhibitor are provided to the patient at the same day, or within a period of no more than 48, 24, 12, 8, 6 or 4 hours.
• the therapy may be an alternating therapy. For example, treatment is started or initiated with a CDC7 inhibitor, followed by treatment with a mTOR inhibitor and/or an immune checkpoint inhibitor.
• treatment may be continued by, for example, treatment with only the CDC7 inhibitor (or, in some embodiments, by e.g. both mTOR and CDC7 inhibitor and/or by both CDC7 inhibitor and immune checkpoint inhibitor or by CDC7 inhibitor, mTOR inhibitor and immune checkpoint inhibitor).
• the steps may be repeated as long as it is to the benefit of the patient.
• a period of treatment (including the initial period) with the CDC7 inhibitor (or, in some embodiments, by e.g. both mTOR and CDC7 inhibitor and/or by both CDC7 inhibitor and immune checkpoint inhibitor or by CDC7 inhibitor, mTOR inhibitor and immune checkpoint inhibitor).
• CDC7 inhibitor may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 .13, 14, 15,
• a treatment with the CDC7 inhibitor is for at least 5 days, for example 5 - 22 days.
• a period of treatment with the mTOR inhibitor may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8 , 9, 10 .13, 14, 15,
• a period of treatment with the mTOR inhibitor is between 2 - 12 days.
• a period of treatment with the mTOR inhibitor is shorter (in days) than a treatment period with the CDC7 inhibitor.
• the CDC7 inhibitor is provided to the patient, no mTOR inhibitor is provided to the patient during that period (or there is an overlap of a short period, for example for one or two days).
• no CDC7 inhibitor is provided to the patient during that period (or there is an overlap of a short period, for example for one or two days).
• a period of treatment with the immune checkpoint inhibitor may be, for example, 1 , 2,
• Treatment with the immune checkpoint inhibitor may, for example, be provided daily of may be provided 2,3 or 4 times a week or by any other treatment schedule commonly used in the clinical practice.
• Suitable examples of treatment regimens according the current invention include: providing the inhibitor of CDC7 and the inhibitor of mTOR and/or the immune checkpoint inhibitor at the same day; providing the CDC7 inhibitor for a period of one week, followed by treatment with the mTOR inhibitor and/or the immune checkpoint inhibitor for a period of one week; providing the CDC7 inhibitor for a period of one week, followed by treatment with the mTOR inhibitor for a period of 3 - 5 days; providing the CDC7 inhibitor for a period of two weeks, followed by treatment with the mTOR inhibitor for a period of two weeks; providing the CDC7 inhibitor for a period of two weeks, followed by treatment with the mTOR inhibitor for a period of 5- 10 days and so on.
• compositions refer to compositions, products, formulation or combinations that are suitable for administration via various routes of administration.
• compositions, formulations, products, and composition according to the disclosure invention normally comprise the drugs (alone or in combination) and one or more suitable pharmaceutically acceptable excipients or carriers.
• combined preparation or “therapeutic combination” “therapeutic pharmaceutical combination” or“combination therapy” as used herein also relates to a "kit of parts" in the sense that the combination partners (a) and (b) can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e. simultaneously or at different time points.
• an effective amount refers to an amount of an agent/pharmaceutical compound required to ameliorate the symptoms of a disease relative to an untreated patient.
• the effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective amount”.
• a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.
• mammal refers to any member of the class Mammalia, including, without limitation, humans and non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
• the term does not denote a particular age or sex. Thus, adult and new-born subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
• the mammal is human.
• pharmaceutically acceptable is employed herein to refer to those combinations as described herein, other drugs or therapeutics, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in human beings and animals, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• “simultaneously” or“simultaneous administration”, as used herein, refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation.
• one drug may be provided orally whereas the other drug may be provided intravenously during a patient’s visit to a hospital.
• the simultaneous, separate or sequential administration of the combination of agents as disclosed herein causes the agents/compounds to be therapeutically active in the patient receiving the treatment, more in particular causes the agents/compounds to be (jointly) therapeutically active in the cancer cells of the patient suffering from the cancer.
• the term "jointly therapeutically active” or “joint therapeutic effect” means that the therapeutic agents of the combination may be given separately (in a chronologically staggered manner, especially a sequence-specific manner) in such time intervals such that in the patient to be treated, the combination still show a (preferably synergistic) interaction (joint therapeutic effect).
• the CDC7 inhibitors for example, by inducing senescence, makes the cancer cells very sensitive to mTOR inhibition.
• the mTOR inhibitor can be provided to the patient as long as cancer cells are still in the state induced by the treatment with the CDC7 inhibitor.
• there is at least one beneficial effect e.g., a mutual enhancing of the effect of the combination partners (a) and (b) as used herein, in particular a synergism, e.g. a more than additive effect, additional advantageous effects, less side effects, a combined therapeutic effect in a non-effective dosage of one or both of the combination partners; very preferably there is a synergism of the combination partners (a) and (b).
• a beneficial effect e.g., a mutual enhancing of the effect of the combination partners (a) and (b) as used herein, in particular a synergism, e.g. a more than additive effect, additional advantageous effects, less side effects, a combined therapeutic effect in a non-effective dosage of one or both of the combination partners; very preferably there is a synergism of the combination partners (a) and (b).
• the present invention relates to a CDC7 inhibitor and a mTOR inhibitor for use in the treatment of cancer in a subject.
• the treatment of cancer in a subject suffering from cancer comprises the step of treating a patient by inhibiting CDC7 activity in the cancer of the patient by providing a CDC7 inhibitor to the patient.
• the CDC7 inhibitor may induced senescence in the cancer cells.
• Non limiting examples of such inhibitors of CDC7 include TAK931 , XL413 and LY3177833 or others mentioned herein.
• the inhibition of CDC7 in the cancer cells is combined with the inhibition of mTOR using an mTOR inhibitor such as, but not limited to AZD8055 or AZD2014 or others as mentioned herein.
• the present inventors have surprisingly found that, by using a CDC7 inhibitor and a mTOR inhibitor in combination (combination therapy) for the treatment of cancer, it leads to an efficient treatment outcome (e.g. great decrease in tumor and/or greater survival rate) relative to a cancer treatment based on monotherapy with a compound capable of inducing senescence in cancer cells (e.g. CDC7 inhibitor compound such as XL413) or relative to treatment with a mTOR inhibitor alone. It was found (without being bound to any theories) that this effect was associated or due to the selective action of mTOR inhibitor compounds on cancer cells that are treated with a CDC7 inhibitor.
• a compound capable of inducing senescence in cancer cells e.g. CDC7 inhibitor compound such as XL413
• mTOR inhibitors can selectively target or act (to kill or inhibit viability) on cancer cells, which were treated with a CDC7 inhibitor (and thereby were induced to undergo senescence (e.g. as indicated by the presence of the marker beta-galactosidase))
• a CDC7 inhibitor and thereby were induced to undergo senescence (e.g. as indicated by the presence of the marker beta-galactosidase)
• the cancer is a cancer (or cancer cell) having a mutated p53 gene or protein.
• the cancer (the cancer subject or cancer cell) has a p53 or a TP53 mutation in his genome leading a loss of wild- type p53 activity (i.e. so-called“loss-of-function” mutation leading to non-functional p53 gene or protein compared to wild type p53 gene or protein) or alterations in wild- type p53 activity (e.g. leading to less functional p53 gene or p53 protein compared to wild type p53 gene or protein) (Muller and Vousden (2014) Cancer cell, Vol. 25, pages 304-317).
• the p53 mutation or TP53 mutation is any mutation that leads to loss-of-function of the p53 gene or p53 protein, i.e. the gene cannot produce a p53 protein or cannot produce a functional p53 protein (has lost biological activity compared to wild type p53 protein) or the p53 protein is altered in such a way that it cannot exert its biological function compared to wild type p53 protein.
• the skilled person is well acquainted with the various type of p53 or TP53 mutation and can determine whether a mutation in the TP53 gene or p53 protein is a loss-of-function mutation (also known as inactivating mutation).
• the mutational status of TP53 gene (or p53 protein) in a subject or a cell e.g. detection of mutant TP53 genes or p53 protein (relative to wild-type TP53 gene or protein) in said subject or cell (e.g. human being such as a cancer patient or a cancer cell), can be detected in tumor samples or, in some types of cancer, in biological samples such as urine, stool, sputum or serum using standard molecular as well as laboratory techniques (e.g. PCR techniques, immunohistochemistry (IHC) techniques on tumor sections stained with a p53 antibody, etc.) to identify mutations and polymorphisms in a gene of interest (e.g. p53).
• the skilled person is well- acquainted with methods for assessing the mutational status of p53 gene or p53 protein (by reference to a wild-type p53 gene or p53 protein) in a sample.
• the CDC7 inhibitor compound may be provided or administered to the subject simultaneously, separately or sequentially with the mTOR inhibitor.
• CDC7 inhibitor compound and the mTOR inhibitor compound as taught herein can be administered to the subject simultaneously, separately or sequentially (as explained herein) using any of the various methods of delivery systems well known to those skilled in the art.. Depending on the type of compounds used, the skilled person knows how to select a suitable delivery system.
• the treatment is initiated by the provision or administration of the CDC7 inhibitor to the subject.
• the treatment is started by providing a CDC7 inhibitor to the subject before a mTOR inhibitor is provided or administrated to the subject.
• the treatment of the patient is initiated/started by providing a CDC7 inhibitor, followed by, for example within several days or weeks as described herein, administration of the mTOR inhibitor (alone or in combination with a CDC7 inhibitor). It is believed that initiating treatment with the CDC7 inhibitor, followed by treatment with the mTOR inhibitor further improves the treatment as this may lead to a situation where a maximal (as much as possible) cancer cells have become sensitive to the mTOR inhibitor (e.g.
• the sequence of events is as follows: that the treatment of cancer (e.g. liver cancer) is initiated or started by first providing or administering the cancer subject (e.g. liver cancer subject) with the CDC7 inhibitor compound (e.g. XL413 or TAK931 or LY3177833).
• the cancer subject which has been treated with the CDC7 inhibitor compound (e.g. XL413 or TAK931 or LY3177833) is then treated with a mTOR inhibitor compound (e.g. AZD8055 or AZD2014).
• the drugs are administered or provided separately or sequentially (one after the other, in any order), it is preferable that the drugs are administered or provided within a certain time limit such that the pharmacological effect of the first drug is still noticeable in the patient, more in particular in the cancer cells in the patient.
• the treatment is simultaneously, i.e. wherein the CDC7 inhibitor and the mTOR inhibitor are provided to the patient at the same day, or within a period of no more than 48, 24, 12, 8, 6 or 4 hours.
• the therapy may be an alternating therapy. For example, treatment is started or initiated with a CDC7 inhibitor, followed by treatment with a mTOR inhibitor.
• a period of treatment with the CDC7 inhibitor may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 .13, 14, 15, .20, 21 ,
• a treatment with the CDC7 inhibitor is for at least 5 days, for example 5 - 22 days.
• a period of treatment with the mTOR inhibitor may be, for example, 1 , 2, 3, 4, 5, 6, 7, 8 , 9, 10 .13, 14, 15, .20, 21 , 22,
• a period of treatment with the mTOR inhibitor is between 2 - 12 days.
• a period of treatment with the mTOR inhibitor is shorter (in days) than a treatment period with the CDC7 inhibitor.
• the CDC7 inhibitor is provided to the patient, no mTOR inhibitor is provided to the patient during that period (or there is an overlap of a short period, for example for one or two days).
• no CDC7 inhibitor is provided to the patient during that period (or there is an overlap of a short period, for example for one or two days).
• Suitable examples of treatment regimens according of the current invention include: providing the inhibitor of CDC7 and the inhibitor of mTOR at the same day; providing the CDC7 inhibitor for a period of one week, followed by treatment with the mTOR inhibitor for a period of one week; providing the CDC7 inhibitor for a period of one week, followed by treatment with the mTOR inhibitor for a period of 3 - 5 days; providing the CDC7 inhibitor for a period of two weeks, followed by treatment with the mTOR inhibitor for a period of two weeks; providing the CDC7 inhibitor for a period of two weeks, followed by treatment with the mTOR inhibitor for a period of 5- 10 days and so on.
• the treatment also comprises short periods (for example 1 , 2, 3, 4, or 5 days) wherein the patient is not treated with a CDC7 inhibitor and not treated with a mTOR inhibitor.
• the cancer may be any cancer, particularly a cancer characterized by the presence of a p53 mutation as taught herein.
• the cancer preferably a cancer characterized by the presence of a p53 mutation as taught herein, is selected from the group of liver cancer, lung cancer such as non-small lung cancer, colon cancer, preferably liver cancer.
• the subject is a human subject (a human cancer subject).
• any CDC7 inhibitor compound may be used.
• the CDC7 inhibitor may be selected from the group consisting of TAK- 931 , XL413, LY3177833, NMS-1 1 16354, and SRA141.
• TAK-931 or TAK931 (Takeda oncology; see also EP 2403857), is an orally bioavailable inhibitor of CDC7. Upon administration, TAK-931 binds to and inhibits CDC7 (Iwai et al (2016) European Journal of Cancer, Vol 69(1) page S34).
• TAK931 can be represented by the following chemical structure:
• the compound XL413 (also known as BMS-863233, from Bristol Myers Squibb (BMS), e.g. see Koltun et al (2012), Bioorg Med Chem Lett., Vol. 22(1 1), pages 3727-31 ; Montagnoli et al (2010) American Association of Cancer Research, Vol. 16(18), pages 4503-4508)is an orally bioavailable inhibitor of CDC7.
• BMS-863233 BMS-863233
• BMS Bristol Myers Squibb
• LY3177833 is an orally bioavailable inhibitor of CDC7. Upon administration, LY3177833 binds to and inhibits CDC7 (see also EP2970221 ). LY3177833 can be represented by the following chemical structure:
• AZD8055 (e.g. see Chresta et al, (2010) Cancer Res., Vol. 70(1), pages 288-98) is an orally bioavailable inhibitor of mTOR.
• AZD-8055 decreases viability of brain tumor cells; in vivo, it inhibits tumor growth.
• AZD-8055 had been in phase I trials by AstraZeneca for the treatment of malignant gliomas and solid tumors. However, this research has been discontinued.
• AZD8055 binds to and inhibits mTOR.
• AZD8055 can be represented by the following chemical structure:
• AZD2014 (also known as Vistusertib, from AstraZeneca ,e.g. see (2015), Guichard et al (2015) Mol Cancer Ther., Vol. 14(1 1), pages 2508-18) is an orally bioavailable inhibitor of mTOR. Upon administration, AZD2014 binds to and inhibits mTOR. AZD2014 can be represented by the following chemical structure:
• any mTOR inhibitor compound may be used, for examples those described herein.
• the mTOR inhibitor inhibits both MTOR 1 and mTOR 2.
• the mTOR inhibitor inhibits mTOR2.
• the skilled person is well aware what, in the context of the current invention, related to the treatment of patients, is an inhibitor of mTOR (or, in case of the CDC7 inhibitor, what is an inhibitor of CDC7).
• the mTOR inhibitor may be selected from the group consisting of AZD8055, AZD2014.
• Non-limiting examples include XL388, GDC- 0349, GSK105965, MLN0128, PI-103, NVP-BEZ235, WJD008, XL765, SF-1 126, Torinl , PP242, PP30, Ku-0063794, WYE-354, WYE-687, WAY-600, INK128, and OSI-027.
• the combination of a CDC7 inhibitor and a mTOR inhibitor may be:
• the CDC7 inhibitor is XL413 and the mTOR inhibitor is AZD8055 or;
• the CDC7 inhibitor is TAK-931 and the mTOR inhibitor is AZD8055 or;
• the CDC7 inhibitor is LY3177833 and the mTOR inhibitor is AZD8055 or;
• the CDC7 inhibitor is LY3177833 and the mTOR inhibitor is AZD2014 or
• the CDC7 inhibitor is TAK-931 and the mTOR inhibitor is AZD2014 or;
• the CDC7 inhibitor is XL413 and the mTOR inhibitor is AZD2014. It was found that these specific combinations of a CDC7 inhibitor and a mTOR inhibitor were particularly effective, i.e. effectively caused growth inhibition of cancer such as e.g. liver cancer (as shown herein, e.g. in vitro). Therefore, in certain embodiments, it may be particularly advantageous to use one of these specific combinations of a CDC7 inhibitor and a mTOR inhibitor for use in the treatment of cancer, e.g. liver cancer, in particular p53 mutated cancers.
• the combination of the CDC7 inhibitor and mTOR inhibitor for use in the treatment of cancer in a subject further comprises the use of an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the present invention relates to a CDC7 inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a mTOR inhibitor, or wherein the treatment further comprises the use of an immune checkpoint inhibitor, or wherein the treatment further comprises the use of an mTOR inhibitor and an immune checkpoint inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the present invention relates to a composition comprising CDC7 inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a mTOR inhibitor, or wherein the treatment further comprises the use of an immune checkpoint inhibitor, or wherein the treatment further comprises the use of an mTOR inhibitor and an immune checkpoint inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014.
• the immune checkpoint inhibitor may be selected as taught herein and is preferably selected from nivolumab, pembrolizumab, BGB-A317, PDR001 , pidilizumab, AMP- 224, AMP-514, cemiplimab, atezolizumab, avelumab, durvalumab, or ipilimumab.
• more than one immune checkpoint inhibitor may be used, for example a PD-1 inhibitor and a CTLA- inhibitor.
• the cancer may be any cancer, preferably liver cancer, and preferably the cancer (the cancer subject or cancer cell) has a p53 or a TP53 mutation in his genome leading a loss of wild-type p53 activity or alterations in wild-type p53 activity (e.g. leading to less functional p53 gene or p53 protein compared to wild type p53 gene or protein).
• the CDC7 inhibitor compound may be provided or administered to the subject simultaneously, separately or sequentially with the mTOR inhibitor as taught herein (by any suitable route of administration as described above), preferably the CDC7 inhibitor compound is provided at initiation of the treatment or administered before the mTOR inhibitor compound as taught herein.
• the CDC7 inhibitor compound may be provided or administered to the subject simultaneously, separately or sequentially with the immune checkpoint inhibitor (or with the immune checkpoint inhibitor and the mTOR inhibitor) as taught herein (by any suitable route of administration as described above).
• the mTOR inhibitor compound when combined in the treatment with a CDC7 inhibitor and an immune checkpoint inhibitor may be provided or administered to the subject simultaneously, separately or sequentially with the immune checkpoint inhibitor as taught herein (by any suitable route of administration as described above).
• the present invention relates to a mTOR inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a CDC7 inhibitor, or wherein the treatment further comprises the use of a CDC7 inhibitor and an immune checkpoint inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4..
• the present invention relates to a composition comprising a mTOR inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a CDC7 inhibitor, or wherein the treatment further comprises the use of a CDC7 inhibitor and an immune checkpoint inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014.
• the immune checkpoint inhibitor may be selected as taught herein and is preferably selected from nivolumab, pembrolizumab, BGB-A317, PDR001 , pidilizumab, AMP- 224, AMP-514, cemiplimab, atezolizumab, avelumab, durvalumab, or ipilimumab. Is some embodiments more than one immune checkpoint inhibitor may be used, for example a PD-1 inhibitor and a CTLA- inhibitor.
• the cancer may be any cancer, preferably liver cancer, and preferably the cancer (the cancer subject or cancer cell) has a p53 or a TP53 mutation in his genome leading a loss of wild-type p53 activity or alterations in wild-type p53 activity (e.g. leading to less functional p53 gene or p53 protein compared to wild type p53 gene or protein).
• the mTOR inhibitor compound may be provided or administered to the subject simultaneously, separately or sequentially with the CDC7 inhibitor as taught herein (by any suitable route of administration as described above), preferably the CDC7 inhibitor compound is provided or administered at initiation of the treatment or before the mTOR inhibitor compound as taught herein.
• the mTOR inhibitor compound when combined in the treatment with a CDC7 inhibitor and an immune checkpoint inhibitor may be provided or administered to the subject simultaneously, separately or sequentially with the immune checkpoint inhibitor as taught herein (by any suitable route of administration as described above).
• the present invention relates to an immune checkpoint inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a CDC7 inhibitor, or wherein the treatment further comprises the use of a CDC7 inhibitor and a mTOR inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the present invention relates to a composition comprising an immune checkpoint inhibitor for use in the treatment of cancer in a subject, wherein the treatment further comprises the use of a CDC7 inhibitor, or wherein the treatment further comprises the use of a CDC7 inhibitor and a mTOR inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD8055, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably XL413, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably TAK-931 , and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014, or
• the CDC7 inhibitor compound may be selected as taught herein, and is preferably LY3177833, and the mTOR inhibitor compound may be selected as taught herein, and is preferably AZD2014.
• the immune checkpoint inhibitor may be selected as taught herein and is preferably selected from nivolumab, pembrolizumab, BGB-A317, PDR001 , pidilizumab, AMP- 224, AMP-514, cemiplimab, atezolizumab, avelumab, durvalumab, or ipilimumab.
• more than one immune checkpoint inhibitor may be used, for example a PD-1 inhibitor and a CTLA- inhibitor.
• the cancer may be any cancer, preferably liver cancer, and preferably the cancer (the cancer subject or cancer cell) has a p53 or a TP53 mutation in his genome leading a loss of wild-type p53 activity or alterations in wild-type p53 activity (e.g. leading to less functional p53 gene or p53 protein compared to wild type p53 gene or protein).
• the immune checkpoint inhibitor compound may be provided or administered to the subject simultaneously, separately or sequentially with the CDC7 inhibitor as taught herein (by any suitable route of administration as described above).
• the immune checkpoint inhibitor when combined in the treatment with a CDC7 inhibitor and a mTOR inhibitor may be provided or administered to the subject simultaneously, separately or sequentially with the mTOR inhibitor as taught herein (by any suitable route of administration as described above).
• the present invention relates to a combination comprising a CDC7 inhibitor, such as XL413, TAK-931 , or LY3177833, and others and a mTOR inhibitor, such as AZD8055, AZD2014, and others, or comprising a CDC7 inhibitor and a mTOR inhibitor and an immune checkpoint inhibitor, and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• a CDC7 inhibitor such as XL413, TAK-931 , or LY3177833, and others
• a mTOR inhibitor such as AZD8055, AZD2014, and others
• the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4.
• the combination comprising a CDC7 inhibitor and a mTOR inhibitor or comprising a CDC7 inhibitor and a mTOR inhibitor and an immune checkpoint inhibitor may be a composition, e.g. a pharmaceutical composition comprising all compounds (compounds are mixed together) or the combination comprising a CDC7 inhibitor and a mTOR inhibitor or comprising a CDC7 inhibitor and a mTOR inhibitor and an immune checkpoint inhibitor may be a composition wherein the CDC7 inhibitor, the mTOR inhibitor and the immune checkpoint inhibitor are provided separately (in different tube or bottle or container) but are mixed before administration to a subject or a provided separately, e.g.
• both compounds one after the other, or sequentially to a subject in a manner that allows both compounds to exert their biological activity or in such a way that both compounds are therapeutically active together (e.g. at some point in time, they overlap in action), in the same cells (e.g. cancer cells).
• the combination as taught herein may be used as a medicament in the treatment of cancer in a subject, more preferably for use in the treatment of cancer in a subject, wherein the cancer (cancer subject or cancer cell) has a mutated p53 gene or expresses a mutant p53 protein or an mRNA encoding a mutant p53 protein, as taught herein.
• the present invention relates to a kit comprising
• container means for containing said first unit dosage form and second unit dosage forms, and optionally, instructions for use; or comprising
• an immune checkpoint inhibitor in a second unit dosage form and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4;
• container means for containing said first unit dosage form and second unit dosage form, and optionally, instructions for use; or comprising
• an immune checkpoint inhibitor is a third unit dosage form and wherein preferably the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4;
• container means for containing said first unit dosage form and second unit dosage form and said third unit dosage form, and optionally, instructions for use.
• the kit may be for use as a medicament, preferably for use as a medicament in the treatment of cancer in a subject, more preferably for use in the treatment of cancer in a subject, wherein the cancer or cancer cell has a mutated p53 gene or expresses a mutant p53 protein or an mRNA encoding a mutant p53 protein, as taught herein.
• the present invention relates to a combination treatment comprising the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor such as XL413, TAK-931 , LY3177833, and others and a mTOR inhibitor such as AZD8055, AZD2014, and others to a subject for use in the treatment of cancer, or comprising the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor and an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4, to a subject for use in the treatment of cancer, or comprising the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor and an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4, and an mTOR inhibitor to a subject for use in the treatment of cancer
• a CDC7 inhibitor such as XL413, TAK-931 , LY3177833, and
• the present invention relates to a method of the treatment of cancer in a subject, wherein the method comprises the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor and an effective amount of a mTOR inhibitor to the subject or wherein the method comprises the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor and an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4, to the subject or wherein the method comprises the simultaneous, separate or sequential administration of an effective amount of a CDC7 inhibitor and a mTOR inhibitor and an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4, to the subject
• the present invention relates to a method for selecting a subject having cancer suitable for simultaneous, separate, or sequential treatment with a CDC7 inhibitor and a mTOR inhibitor, or with a CDC7 inhibitor and an immune checkpoint inhibitor, preferably wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 or CTLA-4, or with a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor, wherein the method comprises the steps of:
• a) Providing a sample comprising cancer cell material from said subject; and b) Detecting in the sample of step a) whether the cancer has a mutated p53 gene or expresses a mutant p53 protein or an mRNA encoding a mutant p53 protein.
• the cancer cell material may be obtained, for example, from a cancer cell biopsy of from a liquid biopsy from blood or other fluid of a cancer patient.
• the sample of step a) has a mutated p53 gene or expresses a mutant p53 protein or an mRNA encoding a mutant p53 protein, it indicates that the cancer subject is suitable for simultaneous, separate, or sequential treatment with a CDC7 inhibitor and a mTOR inhibitor, or with a CDC7 inhibitor and an immune checkpoint inhibitor, or with a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor.
• step a) does not have a mutated p53 gene or does not express a mutant p53 protein or an mRNA encoding a mutant p53 protein, it indicates that the cancer subject is less or not suitable for simultaneous, separate, or sequential treatment with a CDC7 inhibitor and a mTOR inhibitor, or with a CDC7 inhibitor and an immune checkpoint inhibitor, or with a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor.
• the sample comprising cancer cells may be any type samples, such as a tumor tissue sample or tumor biopsy sample, i.e. piece(s) or slice(s) of tissue that has/have been removed from a tumor, including following a surgical tumor resection.
• the sample may be a blood sample comprising cancer cells or urine sample comprising cancer cells.
• the tumor tissue sample or blood or urine sample can be subjected to a variety of well-known post-collection preparative and storage techniques (e.g., fixation, storage of fixed tissue, freezing, homogenization, etc.) prior performing detection of p53 mutation using standard techniques such as immunostaining or prior performing PCR techniques, or in situ hybridization or other laboratory techniques allowing detection of a p53 mutation in said sample.
• step b) it is understood that detecting a p53 mutation performed relative to the p53 wildtype gene or protein.
• the skilled person is well-acquainted with methods for detecting a p53 mutation in a cancer (cancer subject or cancer cell), as taught herein above, and can establish or determine whether a cancer subject is suitable for the combination therapy as taught herein, according to step b) above.
• step b) if the sample of step a) has a mutated p53 gene or expresses a mutant p53 protein or an mRNA encoding a mutant p53 protein, it indicates that the cancer subject is suitable for simultaneous, separate, or sequential treatment with a CDC7 inhibitor such as XL413, TAK-931 , LY3177833, and others and a mTOR inhibitor such as AZD8055, AZD2014, and others, or with a CDC7 inhibitor and an immune checkpoint inhibitor, or with a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor.
• said subject may be treated according to the methods as taught herein, e.g. may be administered with the combination as taught herein.
• the present invention relates to an in vitro or in vivo method for treating cancer cells, the method comprising treating the cancer cells with a CDC7 inhibitor such as XL413, TAK-931 , LY3177833, and others and a mTOR inhibitor such as AZD8055, AZD2014, and others, or with a CDC7 inhibitor and an immune checkpoint inhibitor or with a CDC7 inhibitor, a mTOR inhibitor and an immune checkpoint inhibitor,.
• a CDC7 inhibitor such as XL413, TAK-931 , LY3177833, and others
• a mTOR inhibitor such as AZD8055, AZD2014, and others
• the in vivo method is not performed in a human subject.
• the in vivo method may be performed in any other animal, for example, mammals and rodents including rats and mice.
• human cells e.g. human cancer cells may be used and that have been introduced in a non-human animal (e.g. Patient derived xenografts (model of cancer where the tissue or cells from a patient's tumor are implanted into an (immune-deficient or humanized) mouse.
• FIG. 1 Inhibition of CDC7 as a potential senescence-induction strategy in liver cancer:
• A Schematic outline of the stress lethality screen. Hep3B and Huh7 cells were infected with a lentiviral kinome gRNA library and cultured for 14 days (T14) in replicates. gRNA barcodes in TO and T14 samples were subsequently recovered by PCR and analyzed by next generation sequencing.
• B Pooled gRNA screen identified stress lethal hits in Hep3B and Huh7 cells. Each dot in the plot represents a gRNA from the screen experiment. The y axis shows the fold change in abundance (ratio of gRNA frequency in T14 sample to that in TO sample).
• the x axis represents the frequency (the average counts of sequencing reads in TO sample).
• C Venn diagram of dropout genes (top 50 most strongly deleted genes in each cell line) identified by CRISPR screen in Hep3B and Huh7 cells. Hits with red font are targetable with commercial compounds. Blue font represents that the hits are not targetable with commercial compounds now.
• D Schematic outline of the secondary compound screen. in liver cancer cells (Hep3B and Huh7) and normal cells (BJ and RPE-1) were plated in 96-well plates. For the screen, all compounds were tested at 5 different concentrations for 4 days, and senescence was detected by staining of b- galactosidase activity.
• E Heatmap showing effects of compounds (5 mM) on cell senescence in different cell lines in the secondary screen.
• FIG. 1 Upregulation of CDC7 correlates with poor prognosis of liver cancer patients:
• A Western blot analysis of CDC7, p-MCM2, and MCM2 levels in a panel of normal cell lines and liver cancer cell lines. HSP90 served as a control.
• C Representative images of immunohistochemical staining of CDC7 in liver cancer tissues and the paired noncancerous tissues.
• liver cancer cell lines were measured by western blot.
• B Liver cancer cells (Hep3B, Huh7, SK-Hep1 , and Huh6) were stably infected with pLKO empty vector or with two independent shRNAs targeting CDC7 (shCDC7 #1 , shCDC7 #2). Viability was assessed by colony formation assay. Cells were fixed, stained, and photographed after 10 days of culture.
• C CDC7 knockdown induces senescence in Hep3B and Huh7 cells. Senescence was detected by staining of b- galactosidase activity according to the user manual.
• p53 mutant or null liver cancer cells Hep3B, Huh7, SNU182, SNU398, SNU449, PLC/PRF/5, MHCC97H, and HCCLM3
• p53 wild-type liver cancer cell lines SK-Hep1 and Huh6
• normal cell lines BJ, TIG-3, RPE-1 , and MCF-10A
• GSEA Gene set enrichment analysis
• FIG. 4 LY3177833 and TAK931 in p53 mutant liver cancer cells: (A-B) p53 mutant or null liver cancer cells (Hep3B, Huh7, SNU398, MHCC97H, and HCCLM3) and p53 wild-type liver cancer cell lines (SK-Hep1 and Huh6) were seeded at low confluence and treated with indicated concentrations of LY3177833 or TAK931 twice per week. Viability was assessed by a colony formation assay. Cells were fixed, stained, and photographed after 14 days of culture.
• C-D Liver cancer cells were seeded and culture in presence of indicated concentrations of LY3177833 or TAK931 , the growth curves were measured by IncuCyte® cell proliferation assay.
• E- F Liver cancer cells were cultured in presence of indicated concentrations of LY3177833 or TAK931 for 4 days. Senescence was detected by staining of b- galactosidase activity.
• FIG. 5 mTOR inhibition: (A) the untreated cells were seeded at low confluence, while XL413-treated cells were seeded at high confluence. Then the cells were cultured with different concentration of AZD8055. Cells were fixed, stained, and photographed after 5 days. (B) Control cells and XL413-treated cells were cultured with increasing concentrations of AZD8055 for 96 hr. Apoptotic cells were determined by IncuCyte® caspase-3/7 apoptosis assay according to the user manual. (C) Biochemical responses of Huh7 and Hep3B cells treated with XL413, AZD8055 or their sequential treatment were documented by western blot analysis.
• Control cells and LY3177833-treated or TAK931 -treated cells were cultured with increasing concentrations of AZD2014 for 96 hr.
• Apoptotic cells were determined by IncuCyte® caspase-3/7 apoptosis assay according to the user manual.
• Example 1 Materials Cell lines
• the human liver cancer cell lines (Hep3B, HepG2, PLC/PRF/5, SNU 182, SNU398, SNU449, SK-Hep1 , and Huh6) were purchased from the American Type Culture Collection (ATCC, VA, USA).
• the human liver cell line Huh7 cells was purchased from Riken Cell Bank (Tsukuba, Japan).
• the human hepatocellular carcinoma cell lines MHCC97H and HCCLM3 were provided by the Liver Cancer Institute of Zhongshan Hospital (Shanghai, China).
• hTERT immortalized BJ (fibroblasts) and RPE-1 (retinal pigment epithelial cells) cell lines were provided by Xiaohang Qiao (Amsterdam, The Netherlands).
• MCF-10A non-tumorigenic epithelial cell line
• TIG-3 human lung fibroblast cell line
• Live cancer cells, BJ, and TIG-3 were cultured in DMEM with 10% FBS, glutamine and penicillin/streptomycin (Gibco®) at 37 °C / 5% C02.
• RPE-1 cells were cultured in DMEM: F12 medium with 10% FBS, glutamine and penicillin/streptomycin (Gibco®) at 37 °C / 5% C02.
• AZD8055 (S1555), AZD2014 (S2783), XL413 (S7547), BMS265246 (S2014), ON- 01910 (S1362), BLU9931 (S7819), PD0166285 (S8148), LDC000067 (S7461), PF- 03814735 (S2725), D 4476 (S7642), and VE-821 (S8007) were purchased from Selleck Chemicals.
• THZ531 (A8736) was purchased from ApexBio.
• TAK-931 (CT- TAK931) was purchased from CHEMIETEK.
• LY3177833 (206762) was purchased from MedKoo.
• Antibody against HSP90 was purchased from Santa Cruz Biotechnology. Antibodies against CDC7 (ab10535) , p-MCM2 (S53), (ab109133), and MCM2 (ab4461 ) were from Abeam.
• the kinome gRNA library was introduced into Hep3B and Huh7 cells by lentiviral transduction. Cells stably expressing gRNA were cultured for 14 days. The abundance of each gRNA in the pooled samples was determined by lllumina deep sequencing. gRNAs prioritized for further analysis were selected by the fold depletion of abundance in T14 sample compared with that in TO sample, using methods as described previously (Evers et al (2016), Nat. Biotechnol, Vol. 34(6): 631-633).
• a compound screen including 10 chemical probes that targeting the 14 genes identified by the CRISPR screen was performed. Compounds used for the screen are described in Figure 1 E. Each compound was evaluated in two liver cancer cell lines (Hep3B and Huh7) and two normal cell lines (BJ and RPE-1) using 5 concentrations. The screens were performed in three biological replicates of each line. Senescence associated-p-galactosidase staining was assessed over 4 days after treatment.
• Senescent cells Histochemical Staining Kit (CS0030-1 KT) from Sigma was applied according to the manufacturer’s instructions.
• HCC specimens were obtained from 80 patients who underwent curative surgery in Eastern Hepatobiliary Hospital of the Second Military Medical University in Shanghai, China. Patients were not subjected to any preoperative anti-cancer treatment. Ethical approval was obtained from the Eastern Hepatobiliary Hospital Research Ethics Committee, and written informed consent was obtained from each patient. Overall survival was defined as the length of time between the surgery and death. Patients were censored on the date of the last follow-up or death. Immunohistochemical score was independently assessed by 2 pathologists without knowledge of patient characteristics. The immunostaining score was evaluated on the basis of percentage score x intensity score.
• Indicated cells were cultured and seeded into 96-well plates at a density of 1000- 1500 cells per well. 24 hours later, drugs were added at indicated concentrations. Cells were imaged every 4 hours in IncuCyte ZOOM (Essen Bioscience). Phase- contrast images were collected and analyzed to detect cell proliferation based on cell confluence.
• a stress lethal CRISPR screen combined with a secondary compound screen confirms inhibition of CDC7 as a potential senescence-induction strategy in liver cancer. It has been demonstrated that the CRISPR-Cas9 system can be used in functional genetic screens in the form of pooled guide RNA (gRNA) libraries (Evers et al (2016), Nat Biotechnol, Vol.34(6), pages 631-633).
• gRNA pooled guide RNA
• Senescence associated beta ( -galactosidase (SA ⁇ -gal) staining was assessed 4 days after treatment.
• XL413, a potent ATP-competitive inhibitor of CDC7 kinase was one of the most potent compound in induction of the senescence marker only in liver cancer cells ( Figure 1 E).
• the MCM complex When DNA replication is initiated, the MCM complex is loaded at origins in an inactive form and is then activated during S phase, in a manner that requires both CDC7 and CDK activities.
• the nuclear accumulation of CDC7 in HCC cells is highly suggestive of the dependence of HCC cells on CDC7.
• p53 mutant or null liver cancer cells Hep3B, Huh7, MHCC97H and PLC/PRF/5
• p53 wild-type liver cancer cell lines SK-Hep1 and Huh6
• normal cell lines BJ, TIG-3, RPE-1 , and MCF-10A
• RNAseq analysis in which two different liver cancer cell lines (Hep3B and Huh7) and two normal cell lines (BJ and TIG-3), were treated for 96 hr with 10 mM XL413.
• Gene set enrichment analysis indicated that the genes regulated by XL413 in liver cancer cell lines were enriched for the senescence-associated up- regulated genes (a 77 gene signature in senescent cells, including CDKN1A, CDKN2B, CDKN2D, etc.). Remarkably, we did not observe this enrichment in the normal cell lines (Figure 3F).
• liver cancer cells both p53 mutant and wild-type cell lines
• LY3177833 and TAK931 showed more potent effect in p53 mutant liver cancer cells compared with p53 wild-type liver cancer cell lines ( Figure 4A-D).
• p53 wild-type liver cancer cells were mainly SA-p-gal negative after the treatment ( Figure 4E-F).
• CDC7 inhibition may provide a therapeutic option for senescence-induction in cancer patients, including liver cancer patients with p53 mutation.
• AZD8055 and AZD2014 selectively kills XL413-treated cells, TAK-931 -treated cells and LY3177833-treated cells.
• Huh7 cells were treated with 10 pM XL413 for 5 days and then control cells and XL413-treated cells were plated in 96-well plates.
• XL413-treated cells were treated with 10 pM XL413 for 5 days and then control cells and XL413-treated cells were plated in 96-well plates.
• compounds from GPCR library All compounds were tested at 4 different concentrations. Of the 260 compounds tested, we found a total of 10 compounds could kill the XL413-treated cells at 5 mM. Among these 10 compounds active against XL413-treated cells, 9 compounds also significantly inhibited or prevented survival of untreated Huh7 cells.
• Huh7 cells were injected in nude mice. Upon tumor establishment, xenografts were treated with vehicle, XL413, AZD8055, or combination for about 20 days. As shown in Figure 5F, the combination of XL413 and AZD8055 elicited a potent growth inhibition of Huh7 cells.
• MHCC97H a human HCC cell line with high metastatic potential that was established by the Liver Cancer Institute of Fudan University (Shanghai, China) (Li et al, (2001) World J Gastroenterol, Vol 7, pages 630-636.
• MHCC97H human HCC cell line with high metastatic potential that was established by the Liver Cancer Institute of Fudan University (Shanghai, China) (Li et al, (2001) World J Gastroenterol, Vol 7, pages 630-636.
• MHCC97H human HCC97H cells.
• mice were treated with vehicle, the CDC7-targeted drugs XL413 (100mg kg-1), the mTOR inhibitor AZD8055 (20mg kg-1), or the combination of CDC7 inhibitor plus AZD8055 for 21 days.
• tumors were dissociated as single cell suspension and flow cytometry analyses were performed to determine the content of tumor-associated macrophages (CD45+ CD1 1 b+Ly6C-Ly6G-), CD8 T cells (CD45+ CD3+CD20- NK1.1-CD8+) and CD4 T cells (CD45+ CD3+ CD20- NK1.1-CD4+) relative to total CD45+ leucocytes and cell proliferation (Ki67+) was determined within CD8 T cells and CD4 T cell populations.
• tumor-associated macrophages CD45+ CD1 1 b+Ly6C-Ly6G-
• CD8 T cells CD45+ CD3+CD20- NK1.1-CD8+
• CD4 T cells CD45+ CD3+ CD20- NK1.1-CD4+
• HCC hepatocellular carcinoma
• Checkpoint blockade treatment will be given immediately after HDTV injection, twice a week (100 - 250 microgram CTLA-4, PD-L1 and/or PD1 antibody) for the duration of the treatment.
• CDC7 inhibitor and mTOR inhibitor - see below are provided after HCCs are first visible in MRI, 14-21 days post HDTV injection, in accordance with the present invention.
• Vectors for hydrodynamic tail-vein injection will be prepared using the EndoFree-Maxi Kit (Qiagen) and resuspended in a sterile 0.9% NaCI solution/plasmid mix containing 5 pg of pT3-c- myc (Addgene 92046), 5 pg of pX330-p53 (Addgene 59910) or pX330-Pten (Addgene 59909), and 2.5 pg of CMV-SB13 Transposase. A total volume mix corresponding to 10% of body weight will be injected via lateral tail vein in 5-7 seconds into 6-8 weeks-old females C57BI/6 mice.
• MRI Magnetic resonance Imaging
• TR/TE 2500/25ms
• 32 x 24mm field of view 320 x 240 matrix, resolution of 0.1 mm
• MRI images will be analyzed with MIPAV (Medical, Image, Processing, Analysis, and Visualization software) to calculate tumor volume. Treatment with immune checkpoint inhibitors is as described above.
• MIPAV Medical, Image, Processing, Analysis, and Visualization software
• mice When HCC are first visible by MRI, 14-21 days post HDTVi, tumor size-matched mice will be randomized over the treatment groups: Mice will be dosed 6 days/week with XL413 (100 mg/kg, oral gavage), AZD8055 (20 mg/kg, oral gavage), a drug combination in which XL413 and AZD8055 were administered at the same dose as single agent. For time point analysis mice were sacrificed 14-16 days post-treatment initiation, while for survival curve and endpoint analysis the treatment continued until tumor reached a total volume of 2 cm 3 . No toxicity has been observed over the monotherapy groups.

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